The current project would utilize MLC-2v expression as a genetic marker of the process of ventricular specification during the earliest stages of murine cardiogenesis (i.e., primitive heart tube). The project would integrate unique reagents (antibodies, cDNA probes, expression vectors, cDNA libraries) with in vitro systems (gel shift/mutagenesis/DEPC interference), embryonic stem cell models of ventricular specification, and transgenic model systems. To exact cause-effect relationships between individual factors and ventricular specification, a variety of approaches would be employed to manipulate the expression of specific candidate genes, including microinjection of neutralizing antibodies in cultured ventricular muscle cells, single and double allelic knockouts via homologous recombination in embryonic stem cells, and the generation of transgenic mice which are homozygous for these inactivated alleles of interest by using these ES cells for blastocyst transgenesis. Accordingly, the Specific Aims would be four-fold: 1) To examine the role of a positively acting cis regulatory element (HF-1b) and its corresponding trans-acting factor in the ventricular specification of MLC-2 gene expression during murine cardiogenesis. 2) To examine the role of an adjacent cis regulatory element (HF-1a) and its corresponding trans-acting factor(s) in the process of ventricular specification of MLC-2 expression. 3) To identify the role of a negative regulatory element (HF-3) and its corresponding trans-acting factor(s) in the ventricular specification of MLC-2 expression. 4) To examine the potential interaction of these positive and negative regulatory pathways in the process of ventricular specification.